Development of novel inhibitors specific for human heparanase-1

Article abstract of DOI:10.1246/cl.130231

The octylglycosides 1-3 having heparan sulfate fragments were designed as inhibitors specific for human heparanase-1. Inhibition experiments for the heparanase revealed the inhibitory effects of 1 and 3 (IC50 = 6 and 1.4mM, respectively). It was difficult for compound 2 to inhibit the heparanase activity. Furthermore, the inhibitory action of 3 was specific for the heparanase, whereas 1 and 2 also inhibited the hydrolysis activity of exoenzyme β-glucuronidase from bovine liver.

Full text of DOI:10.1246/cl.130231

doi:10.1246/cl.130231
Published on the web May 29, 2013
797
Development of Novel Inhibitors Specific for Human Heparanase-1
Masashi Ohmae,* Yuki Fujita, Junko Takada, and Shunsaku Kimura
Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510
(Received March 18, 2013; CL-130231; E-mail: ohmae@peptide.polym.kyoto-u.ac.jp)
HO
HO
The octylglycosides 1-3 having heparan sulfate fragments
were designed as inhibitors specific for human heparanase-1.
Inhibition experiments for the heparanase revealed the inhibitory
effects of 1 and 3 (IC50 = 6 and 1.4 mM, respectively). It was
difficult for compound 2 to inhibit the heparanase activity.
Furthermore, the inhibitory action of 3 was specific for the
heparanase, whereas 1 and 2 also inhibited the hydrolysis
activity of exoenzyme ¢-glucuronidase from bovine liver.
O
HO
NH
O(CH2)7CH3
NaO3S
1
COONa
O
HO
HO
O(CH2)7CH3
OH
HO
HO
HO
2
O
Heparan sulfate (HS) is an important bioactive polysaccha-
ride belonging to the glycosaminoglycans (GAGs).1 HS is
composed of alternating (1 ¼ 4)-¡-D-glucosaminide (GlcN)
and hexuronate [(1 ¼ 4)-¢-D-glucuronide (GlcA) and (1 ¼ 4)-
¡-L-iduronide (IdoA)] with O- and N-sulfonato groups in
various proportions.2,3 Such a structurally diverse heteropoly-
saccharide of HS exists ubiquitously on cell surfaces and in
extracellular matrices (ECMs) as a side chain on HS proteogly-
cans (HSPG). HS plays critical roles in tissue morphogenesis
and in the regulation of cytokine functions via the formation of
morphogen gradients and growth factor storage, respectively.4
Human heparanase-1 (Hpa1) is an endo-¢-glucuronidase
categorized in the glycoside hydrolase family 79 (GH79),5
which is secreted in invasive cells such as inflammatory cells
and highly migratory tumor cells.6 Hpa1 specifically hydrolyzes
¢-glucuronide of HS in ECMs and basement membranes to
release or transfer signaling molecules for angiogenesis prereq-
uisite for tumor progression.7,8 Hpa1 is thus involved in the
process of tumor metastasis; therefore, specific inhibitors for
Hpa1 are important for the suppression of tumor progression and
metastasis.2
Hpa1 inhibitors have been pursued actively and are
classified into five groups: naturally occurring heparin and its
derivatives, synthetic polymers mimicking heparin, sulfated
oligosaccharides and their derivatives, putative transition-state
analogues, and other substances such as suramin.2,9,10 In the
present study, we designed Hpa1 inhibitors 1-3, which are
octylglycosides having HS fragments in the category of sulfated
oligosaccharides and their derivatives (Figure 1), and assessed
their inhibitory activities.
COONa
NH
O
O
NaO3S
O(CH2)7CH3
HO
OH
3
Figure 1. Chemical structures of inhibitors 1-3 for Hpa1.
100
80
60
40
20
0
10
102
103
inhibitor / µM
104
Figure 2. Hpa1 inhibitory activities of 1-3: 1, open diamonds;
2, open squares; 3, open triangles.
which is expected to increase the inhibitory activities of 1-3
through hydrophobic interaction with hydrophobic amino acids
in the cleft structure of Hpa1.13-15 The chain length was limited
to C8 in order to avoid in vivo cellular uptake16,17 by the
increased nonspecific affinity for cell membranes. We are
targeting extracellularly released Hpa1.
The inhibitors 1 and 2 were readily prepared in several
chemical reaction steps (see Supporting Information; SI).18 The
disaccharide derivative 3 was newly prepared in eight chemical
reaction steps (also see SI).18
The novel compound 1 and known compound 211 are
monosaccharide derivatives of HS components; in compound 1,
however, the N-sulfonato (NS) group was introduced in the
expectation of a specific affinity for Hpa1, because the GlcNS
structure is found exclusively in HS/heparin in mammalian
tissues. Compound 2 has a ¢-glucuronide structure, which is
involved in the catalysis mechanism of Hpa1. The novel
compound 3 is a disaccharide derivative having a combined
structure of carbohydrate moieties of 1 and 2. The disaccharide
moiety is anticipated to be specific for Hpa1 because it may
escape from binding to exo-type ¢-glucuronidase (EC 3.2.1.31,
GH2).12 An octyl chain was introduced to 1-3 as an aglycone,
Hpa1 inhibition studies were carried out through the SDS-
PAGE colorimetric detection method13 (Figure 2). The inhib-
itory activity of 1 increased gradually with increasing concen-
Chem. Lett. 2013, 42, 797-798
© 2013 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

798
increased affinity of 1 upon introduction of the N-sulfonato
group;22 however, it also inhibited the activity of BLG. The
inhibitor 2 hardly inhibited Hpa1 activity but did inhibit BLG
activity. The disaccharidic inhibitor 3 clearly showed inhibition
for Hpa1. In contrast, the BLG activity was hardly affected by
3. Thus, these results strongly suggest that the disaccharide
structure of 3 is essential for both distinct activity and selectivity
against Hpa1. Further design of HS disaccharide fragments for
Hpa1 inhibitors is now in progress, particularly in terms of the
sulfation patterns3 and the aglycone structure; these species are
expected to exhibit advanced inhibitory activities for Hpa1.
100
80
60
40
20
0
This study was supported by a Grant-in-Aid for Scientific
Research (C) (No. 24550134) from the Japan Society for the
Promotion of Science (JSPS).
0.1
1
10
inhibitor / µM
102 103
104
References and Notes
1
N. Afratis, C. Gialeli, D. Nikitovic, T. Tsegenidis, E.
Karousou, A. D. Theocharis, M. S. Pavão, G. N.
Tzanakakis, N. K. Karamanos, FEBS J. 2012, 279, 1177.
J.-P. Li, Anti-Cancer Agents Med. Chem. 2008, 8, 64.
B. Casu, I. Vlodavsky, R. D. Sanderson, Pathophysiol.
Haemostasis Thromb. 2007, 36, 195.
J. R. Bishop, M. Schuksz, J. D. Esko, Nature 2007, 446, 1030.
B. L. Cantarel, P. M. Coutinho, C. Rancurel, T. Bernard, V.
Lombard, B. Henrissat, Nucleic Acids Res. 2009, 37, D233.
N. Ilan, M. Elkin, I. Vlodavsky, Int. J. Biochem. Cell Biol.
2006, 38, 2018.
Figure 3. Inhibitory activities of 1-3 for BLG determined by
UV absorbance: 1, open diamonds; 2, open squares; 3, open
triangles.
2
3
tration of 1, and the IC50 value was estimated to be 6 mM. In
contrast, the inhibitor 2 did not exhibit inhibition ability for
Hpa1 up to a dosage of several mM, and at 10 mM, weak
inhibition (24%) was observed. The disaccharidic inhibitor 3
showed distinct inhibition activity for Hpa1, as shown by the
sigmoidal dose-response curve.19 The IC50 value of 3 was
estimated to be 1.4 mM.
Mammalian ¢-glucuronidase is widely distributed in tis-
sues, particularly in the liver, spleen, and kidneys. The enzyme
is involved in the metabolism of GAGs in ECMs by cleaving the
¢-glucuronide at the nonreducing terminals.11 Thus, the inhib-
itory activity for this enzyme is one of the unfavorable efficacies
for heparanase inhibitors; we examined the inhibitory activities
of 1-3 for an exo-type enzyme of ¢-glucuronidase from bovine
liver (BLG) by using phenolphthalein ¢-D-glucuronide as a
substrate dye20 (Figure 3).
As we expected, the disaccharidic inhibitor 3 did not exhibit
any significant inhibitory activities for BLG; the inhibitions
were below 10% over a broad concentration range. In contrast,
the inhibitory activities of the monosaccharidic inhibitors 1 and
2 at 4.5 mM increased to 44% and 51%, respectively. The
inhibitor 2 has a terminal ¢-glucuronide structure, which is
required for the enzyme recognition. Therefore, it will inhibit the
enzyme activity competitively. However, the inhibitor 1 has a
completely different structure from that required for recognition
by the enzyme. Therefore, it was very surprising that 1 exhibited
inhibitory activity for BLG. BLG has been reported to bind to
lactose and N-acetyllactosamine,21 which are not substrates for
the enzyme catalysis; therefore, the inhibitor 1 may also bind the
enzyme via an unknown mechanism. In any case, it is necessary
to examine the mechanism of action of 1 for the activity of BLG
in future.
4
5
6
7
8
9
P. Carmeliet, P. K. Jain, Nature 2000, 407, 249.
G. Bergers, L. E. Benjamin, Nat. Rev. Cancer 2003, 3, 401.
V. Ferro, E. Hammond, J. K. Fairweather, Mini-Rev. Med.
Chem. 2004, 4, 693.
10 S. Cochran, C. Li, J. K. Fairweather, W. C. Kett, D. R.
Coombe, V. Ferro, J. Med. Chem. 2003, 46, 4601.
11 N. Ferlin, D. Grassi, C. Ojeda, M. J. L. Castro, E. Grand, A. F.
Cirelli, J. Kovensky, Carbohydr. Res. 2008, 343, 839.
12 K. Paigen, Prog. Nucleic Acid Res. Mol. Biol. 1989, 37, 155.
13 K. Ishida, G. Hirai, K. Murakami, T. Teruya, S. Simizu, M.
Sodeoka, H. Osada, Mol. Cancer Ther. 2004, 3, 1069.
14 T. Karoli, L. Liu, J. K. Fairweather, E. Hammond, C. P. Li, S.
Cochran, K. Bergefall, E. Trybala, R. S. Addison, V. Ferro,
J. Med. Chem. 2005, 48, 8229.
15 K. Dredge, E. Hammond, K. Davis, C. P. Li, L. Liu, K.
Johnstone, P. Handley, N. Wimmer, T. J. Gonda, A. Gautam,
V. Ferro, I. Bytheway, Invest. New Drugs 2010, 28, 276.
16 H. Nakajima, Y. Miura, T. Yamagata, J. Biochem. 1998, 124,
148.
17 T. Sato, K. Hatanaka, H. Hashimoto, T. Yamagata, Trends
Glycosci. Glycotechnol. 2007, 19, 1.
18 Supporting Information is available electronically on the CSJ-
Journal Web site, http://www.csj.jp/journals/chem-lett/index.
html.
19 R. D. Blumenthal, D. M. Goldenberg, Mol. Biotechnol. 2007,
35, 185.
20 J. Combie, J. W. Blake, T. E. Nugent, T. Tobin, Clin. Chem.
1982, 28, 83.
In the present study, three kinds of the octylglycosides
having HS fragment structures were prepared and evaluated as
inhibitors for Hpa1. The inhibitor 1 showed a very weak but
distinct inhibitory activity for Hpa1, although 1 does not include
the ¢-glucuronide structure. This is probably due to the
21 H. Matsushita-Oikawa, M. Komatsu, N. Iida-Tanaka, H.
Sakagami, T. Kanamori, I. Matsumoto, N. Seno, H. Ogawa,
Glycobiology 2006, 16, 891.
22 S. Peterson, J. Liu, J. Biol. Chem. 2012, 287, 34836.
Chem. Lett. 2013, 42, 797-798
© 2013 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/